Listeriosis is a severe food-borne disease caused by the bacterium Listeria monocytogenes and is a significant public health concern. There are an estimated 1600 cases of Listeriosis per year in the US resulting in 250 deaths. In addition to this high mortality rate infection during pregnancy can lead to miscarriage or stillbirth. Early detection of the pathogen in the food supply is key to protecting public health. Repeated outbreaks of Listeriosis in recent years highlight the need for development of a rapid, sensitive and inexpensive biosensor. In addition, treatment of infected pregnant women requires a course of highly invasive intravenously administered antibiotics. An inexpensive and effective prophylactic that could prevent Listeria colonization would be a powerful addition to prenatal care. We propose using nanobodies to address these critical holes in the defense against Listeriosis. Nanobodies are heavy chain, single domain antibodies derived from the unique immune system repertoires found in Camelidae species (camels, alpacas, llamas). Nanobodies are inexpensive to produce, highly stable, and have distinctive binding modes making them uniquely suited to developing new Listeria biosensors and therapeutics. The goal of this project is to understand how nanobodies derived from a nave Camelidae phage display library bind and interact with Listeria surface antigens, to lay the foundation for development of novel Listeria biosensors and therapeutics. Our preliminary studies have identified four nanobodies that bind to a target that is both surface exposed and required for Listeria virulence factor. Usin structural biology and binding studies we will assess the range of epitopes recognized by nanobodies derived from a combined nave Camelidae phage display library to Listeria virulence factor InlB (AIM 1). We will also employ microbiological and structural studies to examine the potential of Camelidae nanobodies to detect and neutralize Listeria (AIM 2).